Challenge: Developments of enabling technologies underpin continuing advances in biomolecular research. For instance, mass spectrometry (MS)-based sequencing techniques have spurned proteomics research in the past decade. Currently, there is no gold standard technique in metabolomics that allows a routine characterization of the thousands of constituents contained in these samples. NMR is limited to the more abundant analytes due to sensitivity issues. On the other hand, MS is typically capable of detecting many more features, but is often not able to structurally characterize these molecules. Rationale: By coupling tunable infrared (IR) lasers to mass spectrometry instrumentation, the IR spectra of mass- separated ions can be recorded. IR laser spectroscopy of ions combines the high sensitivity and ability to analyze complex mixtures of MS with the enhanced structural information from vibrational spectroscopy. The technique hence allows a chemical elucidation of many unknowns based on diagnostic vibrations and IR spectral fingerprints. Aim 1: Development of cryogenic mass spectrometry and multiplexed IR spectroscopy. In order to make IR spectroscopy a useful bioanalytical tool for biomolecular ions, it is essential that the IR spectra of analytes are well- resolved, and thus distinguishable, and that multiple analytes in mixtures can be probed simultaneously in a multiplexed fashion. We propose to develop a custom-built, cryogenic linear ion trap, where the ions are tagged with weakly-bound molecules (e.g. N2), which are selectively detached upon resonant IR absorption. Aim 2: IR spectroscopy of mass-separated metabolites. Our application of IR spectroscopy of biomolecules focuses on metabolites, where we expect the technique to have most potential. Control experiments on standard metabolites will establish how many analytes can be successfully probed in a multiplexed approach. The methodology will then be applied to selected metabolite samples from colon cancer studies, which have previously been analyzed by high- throughput liquid chromatography and high-resolution mass spectrometry. Aim 3: Structural elucidation of unknown metabolites by comparison to computed IR spectra and bioinformatics approaches. The ultimate goal of this proposal is to chemically characterize unknown biomarkers that cannot be identified by current MS approaches. This requires a comparison of the experimental data for each analyte, namely its mass and its IR spectrum, to putative matches from metabolite databases. The IR spectra of known standards (from aim 2) will serve as a training set and as a benchmark for implementing this identification methodology. Innovation and Impact: The techniques developed here are expected to have the largest impact in global metabolomics, where current tandem mass spectrometry methodologies limit the number of constituents that can be identified in these mixtures. We expect the enhanced structural information from vibrational spectroscopy to yield many new insights in biomarker discovery.